Dimethyl sulfate and diisopropyl sulfate as practical and versatile O-sulfation reagents

O-Sulfation is a vital post-translational modification in bioactive molecules, yet there are significant challenges with their synthesis. Dialkyl sulfates, such as dimethyl sulfate and diisopropyl sulfate are commonly used as alkylation agents in alkaline conditions, and result in the formation of sulfate byproducts. We report herein a general and robust approach to O-sulfation by harnessing the tunable reactivity of dimethyl sulfate or diisopropyl sulfate under tetrabutylammonium bisulfate activation. The versatility of this O-sulfation protocol is interrogated with a diverse range of alcohols, phenols and N-OH compounds, including carbohydrates, amino acids and natural products. The enhanced electrophilicity of the sulfur atom in dialkyl sulfates, facilitated by the interaction with bisulfate anion (HSO4-), accounts for this pioneering chemical reactivity. We envision that our method will be useful for application in the comprehension of biological functions and discovery of drugs.

The submitted paper deals with a pioneering method for O-sulfation, a crucial posttranslational modification and biotransformation within the metabolism of bioactive molecules.The introduction begins by emphasizing the significance of O-sulfation in various biomolecules and highlights the challenges in their synthesis.Traditional methods, such as sulfur trioxide-nitrogen base complexes, face limitations, prompting the need for alternative strategies like early-stage sulfation.The authors introduce dialkyl sulfates, specifically dimethyl sulfate (DMS) and diisopropyl sulfate (DPS), as promising sulfate diesters for Osulfation.
The proposed method involves activating DMS or DPS under tetrabutylammonium bisulfate, enhancing their reactivity for efficient sulfate transfer.Extensive optimization studies identified DMS as the optimal sulfate source, coupled with Bu4NHSO4 as the ideal activation agent.Control experiments validated the necessity of both DMS and Bu4NHSO4 for successful sulfation.The method demonstrates versatility, effectively sulfating a range of primary and secondary alcohols with DMS and DPS, respectively.
The authors showcase the broad applicability of their method to various compounds, including primary alcohols, secondary alcohols, amides, halides, nitrates, ethers, alkenes, alkynes, boronic esters, aldehydes, sulfones, esters, ketones, sulfonamides, aromatics, and heterocycles.Additionally, the method proves effective for the gram-scale synthesis of sulfated products.
The paper extends the method's application to carbohydrates, amino acids, and steroids.Various sugar derivatives, amino acids, and complex natural products were successfully sulfated under mild conditions, showcasing the method's practicality for late-stage modifications of bioactive molecules.
Results from control experiments provide insights into the reaction mechanism, indicating the involvement of key intermediates such as methyl monosulfates and methyl sulfate D'.Notably, the review includes 18O-labeling experiments, confirming the formation of O-SO3 bonds in the sulfation process.Of note, the manuscript is accompanied by extensive supplementary information (122 pages) In conclusion, the paper preents the discovery of a novel activation method for dimethyl sulfate and diisopropyl sulfate as a significant advancement in the synthesis of organic sulfates.The method's mild reaction conditions, broad functional group tolerance, and applicability to complex biomolecules position it as a powerful tool for late-stage sulfation and drug discovery.The authors anticipate the widespread use of this O-sulfation method in comprehending biological functions and advancing drug discovery.
There are no major problems in the manuscript, just several minor comments can be found in the annotated manuscript and supplementary file.

Reviewer #3 (Remarks to the Author):
The manuscript by Yue et al. represents a noteworthy progression in the field of O-sulfation chemistry.Notably, the authors have elucidated a novel reactivity exhibited by dimethyl sulfate and diisopropyl sulfate, serving as innovative O-sulfation reagents under acidic conditions.The transformations outlined predominantly involve the O-sulfation of alcohols, phenols, and carbohydrates, resulting in the production of sulfonated chemicals.This research is particularly intriguing as it refines the reactivity of dimethyl sulfate and diisopropyl sulfate, traditionally acknowledged for their robust alkylating properties, by harnessing their potential as sulfation reagents.
The authors demonstrate the method work with a broad array of electronically and sterically diverse alcohols, carbohydrates, amino acids, and natural products, especially for showcasing selectivity on substrates (55-58).It is noteworthy, however, that the method exhibits limitations in the sulfation of tertiary alcohols and phenols when employing DMS or DPS as the reagents.The authors, in an attempt to address the sulfation of phenols, utilized sodium pyrosulfate as a reagent, a tangent that is not directly pertinent to the focus of this manuscript.Nevertheless, this limitation does not diminish the significance of the work, as the manuscript furnishes compelling evidence that dimethyl sulfate and diisopropyl sulfate can indeed serve as O-sulfation reagents for alcohols and carbohydrates, exhibiting a novel reactivity.
The elucidation of how the reactivity of dimethyl sulfate was modulated presents an intriguing aspect of this study.While the paper does furnish some evidence for the proposed pathway, there remain substantial mechanistic inquiries that warrant attention, particularly concerning the substantiation of the reactive intermediate.Several mechanistic hypotheses pivot on the existence of methyl monosulfates (1-B).As it currently stands, additional experiments are imperative to offer more compelling evidence regarding its presence.Given the substantial emphasis on mechanistic elucidation within this manuscript, these unresolved issues pose impediments to recommending it for publication.
In summary, this work presented by Li and their colleagues represents an important contribution to the field, showcasing a significant advancement in O-sulfation chemistry.The broad substrate scope and excellent selectivity observed in this study open up new avenues for the synthesis of complex and valuable molecules.The compounds are appropriately characterized, and manuscript was well-written.In my opinion, the manuscript merits acceptance for publication in Nature Communications, pending the authors' careful attention to the following suggestions raised.
Conditions optimization: 1.Given the author's emphasis on the importance of acidic conditions in this reaction, have the authors attempted to incorporate the corresponding acid for entries 5-6 in Table 1? 2. On the other hand, the yield with a counter anion other than HSO4 is quite good for entries 4-6, especially for entry 5, even in the absence of acid.Does the author have an explanation for how the product was formed in this case?Substrates scope: 3.All the products were well-characterized; however, only NMR yield was provided for substrates (11, 19-24, 58, 61, 65, 66). 4. For conditions d in Table 2, several other counter cations like Na+, K+, were used.How did the authors determine that these were not included in the final products?Mechanistic studies: 5.In Figure 2 A, the 1H NMR spectrum of methyl monosulfate (1-B) does not appear consistent with it being the main intermediate, especially given its low integration.Without integrations of the key signals, it is hard to discern the correlation between the new peaks in the 1H NMR and that they correlate to the main intermediate.Integration of these peaks in relation to the standard in 1H spectra would be helpful.6.According to the proposed mechanism in Figure 1C-ii, Bu4NHSO4 would react with intermediate B. Why wouldn't it react with DMS at the beginning, given that DMS is less sterically hindered than intermediate B? 7. Have the authors investigated the formation of SO3 during the reaction?They could attempt to detect and capture SO3 by mixing the DMS and Bu4NHSO4.This would enhance the understanding of the reaction mechanism.8. Determining the source of the 'sulfur' in the product is crucial, and conducting experiments with 34S labeled Bu4NHSO4 and/or DMS would provide valuable insights.9.In drawing the mechanism (Figure 1C-ii, from A to B), based on the arrows the authors have depicted, it suggests the formation of a methoxide anion.However, considering the acidic conditions, the existence of a methoxide anion is improbable.The arrow might be better represented as originating from the oxygen of the alcohol rather than the hydrogen.SI and others: 10.It appears that the Rf values for most products are identical.Could the authors please confirm this observation?Additionally, it's noted that most of the products share the same physical state, described as a light yellow oil.11.The abbreviation for diisopropyl sulfate is not consistent between the main text and the SI.Please ensure uniformity in the usage of abbreviations throughout the manuscript.12.Some typos: Figure 1C-Ii -> Figure 1C-ii; line 93: 12 h -> 12 hours; lines 95-98: entry -> entries; line 97: slower yield -> lower yield…

Referees' comments:
Reviewer 1 (Remarks to the Author): We would like to thank the reviewer for their time and consideration of this manuscript.We are grateful for their advice/corrections to improve the quality of our initial submission.Please find below, a list of comments, responses, and the changes made to the manuscript and/or supplementary information.
Yue and colleagues discuss the use of dimethyl and diisopropyl sulfate as a new sulfation reagent.The work is generally well-performed and sufficient in scope and novelty to warrant publication.
We are grateful to the reviewer for this comment.
However, I have some concerns with the paper: 1) What advantage does the reagent have over other best in class sulfation agents?Already known to some extent eg amine-SO3 reagents.We thank the reviewer to pointing this out.To best of our knowledge, other classic sulfation agents include sulfuryl chloride, SO3-amide (DMF) complex and sulfuryl imidazolium salts.With their increased stability, readily availability and straight maneuverability, DMS and DPS have shown a broad substrate scope and wide functional group tolerance, spaning from carbohydrates, amino acids to natural products.
2) The reagent is known to be horribly toxic and mutagenic-this needs to be dealt with in the paper and SI.We appreciate the reviewer's concern for hazard statements of DMS and DPS.

The safety warning for DMS and DPS has been added in the revised manuscript at Table 2's caption:
"Note: The liquid DMS and DPS are volatile and toxic.Exercise extreme caution when handling the liquid."Supporting Information (page 5): "Note: Alkyl sulfate is highly toxic, so the reaction process should be handled with care.It is recommended to work in a fume hood." 3) Has the use of the CF3 derivative of the reagent been considered (less toxic) We appreciate the reviewer's suggestion to explore DMS fluoride derivatives as potential sulfation reagents, as they could potentially offer a safer and more efficient alternative.However, it is important to note that the synthesis of bis(trifluoromethy) sulfate needs the use of fluorine gas, which is an exceedingly complex process, with only 0.1% yield (Journal of the Chemical Society, Perkin Transactions 1, 1979, 2675-2678).Even if practical synthesis methods were to be developed, it is reasonable to doubt whether dimethyl sulfate alone can effectively carry out the sulfation process.The reaction mechanism from dimethyl sulfate suggests that the released HOCF3 is highly unstable and prone to decomposition into fluorophosgene III and fluoride.As a result, alcohol substrate would likely react with fluorophosgene III to form the corresponding fluoroformate IV, which would then be susceptible to attack from any nucleophile present in the reaction mixture, ultimately leading to sulfation failure.Furthermore, our attempts to synthesize DPS fluoride derivatives were not successful under the various conditions examined.
Conversely, diaryl sulfates can be easily obtained; however, they exhibit a lack of reactivity due to their high stability.
These results are included in the SI (page 6): 4) If the bisulfate acts as protic catalysis why is it so special over a simple protonation method?We greatly appreciate the reviewer for highlighting the crucial role of bisulfate in the sulfation process.HSO4 - plays two essential functions in sulfation: 1. Activation of DMS or DPS; 2. Nucleophilic cleavage of the intermediate 1-B.After activation of dialkyl sulfations by protic acids with other anions (HX), the nucleophilic X -may also cleave the intermediate methyl monosulfates 1-B, and generate the product C as well as the methyl derivatives D-1.Among these compounds, only D (X = HSO4 -) can proceed with sulfation further.In contrast, sulfation will be halted at this step without the presence of HSO4 -.Therefore, the special role of bisulfate lies in acting as both a proton acid activator and a source of sulfate.

5) Has N or S sulfation been tested to show generality?
We agree that it would be appropriate to further extend the substrate scope.In response to this feedback, we have tested six substrates, two of which are thiol and thiophenol, affording S-sulfated products.Unfortunately, our method is not suitable to N-sulfation.This is because the protonation of N leads to the formation of ammonium or iminium salts, which cannot undergo nucleophilic attack on DMS or DPS.
In the revised manuscript: "This new protocol was also successful in producing S-sulfation products 20 and 45 for thiol and thiophenol.However, nitrogen compounds such as amine and imine (Supplementary Table 4) did not yield any desired sulfation products, and all starting materials were recovered." The Table 2 was updated as below, and new data can be found in revised SI (page 17, 30). 1 what is the solubility profile of KHSO4 in MeCN -could this be a reason?We agree with the reviewer's observation that the poor solubility of KHSO4 in MeCN is responsible for its failure in sulfation.Less than 1 mg (0.6 mg) of inorganic salt KHSO4 could be dissolved soluble in MeCN (1 mL) at 80 o C: to a 4-mL borosilicate vial was added KHSO4 (32.7 mg) and MeCN (1.0 mL).After heating to 80 °C, the hot mixture was filtered, and residue solid was then dried under vacuum, resulting in the recovery of KHSO4 (32.1 mg).Moreover, the combined use of Bu4NBF4 with KHSO4 proved to be effective in this transformation due to the increased solubility of the activator (HSO4 -).7) Have other alkyl sulfates been tested as comparitors to Bu4NHSO4?We thank you the reviewer for this suggestion.We have attempted several tetra-alkyl ammonium bisulfates (from C1 to C6), all of which delivered the desired product in 70% to 84% yield.Although the reasons for the differences in yield between these alkyl substituents are unclear, we suspect that the solubility and stability of the corresponding organosulfate ammounium salt may play a role, as the n Bu can ensure product stability while increasing solubility (Chem.Commun.2019, 55, 4319-4322).These results can be found in revised Supporting Information (page 6):

6) In Table
8) The work of Kowalska 2012 Kowalska, J., Osowniak, A., Zuberek, J. & Jemielity, J. Synthesis of nucleoside phophosulfates.Bioorg.Med.Chem.Lett.22, 3661-3664 (2012).Should be considered as has a bearing on the work proposed We added this new reference as No. 46, which is about nucleophile of bis(tributylammonium) sulfate.Therefore, we reorganized the numbering of the reference.In the revised manuscript: "These outcome confirmed the crucial role of Bu4NHSO4 for enhanced solubility of sulfate product 1 as tetrabutylammonium salt as well as facile removal of the methyl group in sulfate monoester B. 46 " "Treatment of methyl monosulfates (1-B) with tetrabutylammonium bisulfate (Bu4NHSO4) or tetrabutylammonium acetate (Bu4NOAc) resulted in the formation of the sulfated product 1 with high yields, indicating both bisulfate (HSO4 -) and acetate (AcO -) are effective nucleophilile to cleavage the methyl protecting group in monosulfates 1-B (Figure 2B). 46" 9) Why is reaction not at bp of MeCN -80 not 82 C? There is no difference in our reaction between 80 or 82 °C.We specifically selected 80 °C based on safety considerations.By conducting the reaction slightly below the MeCN boiling point (82 °C), we can avoid reflux, which is both favorable for the reaction and minimizes any potential hazards.
10) Do the arylsulfates undergo rearrangement to the phenol and C-sulfonates?
We appreciate the reviewer's concern about the rearrangement.Under our reaction conditions, no rearrangement of arylsulfates occurred.As an example, we randomly selected substrate 38, which only gives phenol arisen from the hydrolysis in 26% yield under our standard conditions.11) Why is the selection of DPS over DMS justified -unclear to me why one used over another?We thank the reviewer for pointing this out.We specifically chose to study DMS as a novel concept to demonstrate that DMS can function not only as a well-known methylating reagent, but also as a sulfating reagent.For practical purposes, DPS exhibits higher reactivity than DMS for most substrates, including primary and secondary alcohols.Moreover, it is less toxic with its larger molecule weight and higher boiling point.
12) Why is the need for Na2S2O7 required in some examples -mechanistic purpose?We regret that we were not able to articulate clearly in our original submission.As we originally mentioned, phenols, tertiary alcohols, and N-OH compounds are generally considered to have weaker nucleophilicity compared to primary and secondary alcohols.This makes them less reactive and sluggish towards attacking dialkyl sulfates, even with acid activation.On the other hand, Na2S2O7, on the other hand, possesses a stronger electrophilic sulfur atom (as a form of "anhydride" of sodium sulfate) and a better leaving group (SO4 2-).
These unique properties makes it more favorable for the sulfation to occur, especially when dealing with challenging substrates.The mechanistic purpose was added in revised SI (page 7): 13) With more hindered alcohols why does the more hindered DPS work better than DMS?We thank the reviewer for pointing this out.Based on the proposed mechanism in Figure 1C-ii, there may be two factors leading to the different reactivity between DMS and DPS: 1.The steric hindrance between A and substrate ROH.
2. The leaving group ( i PrO -vs MeO -) at step A→B.
3. Stability of carboncation ( i Pr + vs Me + ) at the step B→C; We agree with the reviewer that the larger group of i Pr in DPS is not benefit for the nucleophilic attack of hindered alcohols.However, the better leaving group of i PrO (pKa: i PrOH 29.0, MeOH 30.3;JOC, 1980, 45,  3295-3299) and the greater stability of i Pr are facile to the sulfation.The reaction may be primarily influenced by the latter two factors.
14) Any selectivity between primary/secondary/tertiary alcohols?(not phenol) We thank the reviewer for additional discussion on the selective sulfation among primary, secondary, tertiary alcohols.We agree with the reviewer for more discussion on the selective sulfation.As demonstrated in the manuscript, we were able to achieve selectivity between primary and secondary alcohols (57-59), and found that steric effects play a significant role in determining the selectivity.Now, we further investigated the selectivity between primary and tertiary alcohols (60).However, we acknowledge that achieving selectivity among substrates with similar steric hindrance has been more challenging.In the case of compound 71s with two secondary hydroxyl groups, both can be sulfated.We would like to thank the reviewer for bringing attention to these points and we will continue our efforts to advance selectivity in sulfation reactions.
In the revised manuscript, we add more discussion on the selectivity: "The selectivity in these reactions appears to be mainly controlled by steric effects, as no distinction was observed among substrates with similar hindrance (see Supplementary Information)." The new data for sulfation on substrate 71s, are included in the revised supplementary information (page 40).15) Would the 18 O experiment be better on the DMS? Eg could the sulfate not come from Bu4NHSO4 a bit like that Bu3NSO3 reagent from a few years ago?We agree with the reviewer that the 18 O-DMS is a direct method to determine the source of sulfate.However, there are two types of 18 O-labeled DMS depending on oxygen location: 18 O-DMS 1 and 18 O-DMS 2.
The synthesis of type 1 from Me 18 OH may be not difficult, but it will be cleaved by substrate ROH and exchanged with Bu4NHSO4 during the reaction, providing no hints for our mechanism investigation.However, the synthetic route to 2 is unknown, regardless of whether it is from 18 O2 or H2 18 O.We are unsure if we can complete this complicated and time-consuming procedure.On the contrary, we have more evidence to support the source of sulfate.Please see our reply to comment 8 of reviewer 3 for more details.
LC-MS of the above mixture: 110.9757 was HRMS of the methyl sulfate (MeOSO3 -), no information was found for Bu3N•SO3 (exact mass: 265.1712).
GC-MS of the above mixture: no mass signal was found for SO3 (m/z = 79.9568).
In the revised manuscript, we add: "DMS reacts with tetrabutylammonium bisulfate (Bu4NHSO4) to yield a mixture of D and D' (Figure 2B) rather than SO3 complex, which substantiates the possible path b in the reaction (Figure 1C-ii)."16) Didnt follow this sentence "outcome clearly demonstrated that the sulfation proceeds via the formation 179 of O-SO3 bond rather than C-OSO3, as evidenced by the" was unclear as the level of O18 incorporation is not specified or data on isotopic MS in place?Please see our reply to the review's following comment 3 on Supporting Information.17) Recent work by Lara Malins and others to be cited on sulfating strategies is lacking.We regret that we did not include this literature in our original submission.We have added the suggested references (No. 30) to the second paragraph of the introduction: "Sulfur trioxide-nitrogen base complexes are the most commonly used reagents for sulfating various molecular motifs containing alcoholic, phenolic, amino, thiol and other functional groups. 27-30" Supporting Information: 1) Why does 11, 19, 58, 65 give low conversion/unpurifiable? Needs to be clarified in text too.We thank the reviewer for pointing this out.These four substrates can all be purified, allowing us to include NMR and HRMS data in the original supplementary information.We preferred their yields determined by 1 H NMR over the isolated yields because of the significant differences between them, which were caused by their decomposition on chromatography.As a response to similar comments raised by reviewer 3, both the purified compound yields and the yields determined by NMR are reflected in the new table, and the new data are included in the revised supplementary information.Compound 11: revised yield is 70% b (92% c ), previously 92% b,c .Compound 19: revised yield is 24% b (34% c ), previously 34% b,c .Compound 24 (renumbered 25): revised yield is 75% b (94% c ), previously 94% b, .Compound 27 (renumbered 28): revised yield is 21% b (33% c ), previously 33% b,c .Compound 28 (renumbered 29): revised yield is 29% b (40% c ), previously 40% b,c .Compound 58 (renumbered 61): revised yield is 70% e (88% f ), previously 88% e,f .Compound 61 (renumbered 64): revised yield is 45% a (60% f ), previously 60% a,f .Compound 65 (renumbered 68): revised yield is 71% a (90% f ), previously 90% a,f .Compound 66 (renumbered 69): revised yield is 38% e (49% f ), previously 49% e,f .
In the revised manuscript, we add: "Due to the lability of organic sulfates on chromatography, such as 11, 19 and 25, the purified compound yields was significantly lower than the yields determined by 1 H NMR." 2) 18O experiment the quench should be incorporated not 16OH2 We thank the reviewer for pointing this out.To prevent contamination from 16 O, we have re-run the reaction without the quenching step.We have added this new procedure in the revised SI (page 49): "After stirring for 12 h at 80 °C, the reaction was cooled down to room temperature, and concentrated by rotary evaporation.………" 3) 18O % incorporation not given -essential.We agree with the reviewer's assertion that the incorporation of 18 O in final is essential for reaction mechanism.As a result, we have made significant updates to ensure that both the starting material and the resulting product possess a nearly identical enrichment of 18 O.The data presented in our study demonstrates that no erosion of 18 O occurs, thereby supporting the notion of an intact C-O bond throughout the entire reaction process.
Another solid evidence is that various chiral substrates (46-59, 67-69) proceed with stereoselective retention.Most importantly, R-SH substrates affords S-SO3 products 20 and 45 without formation of O-SO3.The intact C-S bond clearly demonstrated that the sulfation of R-OH proceeds via the formation of O-SO3 bond rather than C-OSO3, as the C-S bond is much weaker than C-O.
We have indicated this result in the revised manuscript (Figure 2F): "To further elucidate the reaction pathway and the source of oxygen in the final product, we conducted 18 Olabeling experiment with compound 70a, which resulted in the successful isolation of alcohol 18 O-70 in 46% yield with a nearly identical level of 18  We thank the reviewer for pointing this out.The presence of a small impure substrate 36 (updated No. 37) led to 2 signal peaks in 19 F-NMR.We have re-run the reaction for compounds 37.The yields of the purified compounds are reflected in the new Table 2, and the new NMR spectra are included in the revised supplementary information (page 98).Compound 37: revised yield is 90% (previously 91% yield).7) NMR for 58 has a drift on baseline why?
The drift on baseline is probably caused by shimming from the instrument, or the structure of 58 containing a phenol group.After Na + exchange, the sodium sulfate 58 (updated No. 61) with longer acquisition times and more delay time to relax between pulses can quiet down the baseline and allow for better phasing.The new NMR spectra is included in the revised supplementary information (page 122).

8) Which compounds are NOVEL?
In the SI, all the new compounds have been thoroughly characterized by 1 H NMR, 13 C NMR and HRMS.Additionally, the known compounds (24, 40 and 42) have been characterized alongside appropriate references.Regarding the reviewer's comment about "NEVEL", it might refer to the novelty of the structure, indicating that it has not been previously reported.In our substrate table, the N-OSO3 (31, 32) and tertiary sulfate (30) compounds are novel molecules that are not otherwise readily accessible.

Reviewer 2 (Remarks to the Author):
We appreciate the reviewer's meticulous analysis of our work, and their advice/corrections to improve the quality of our initial submission.We agree with all comments made by this reviewer and present here the changes we made to the revised submission.
The submitted paper deals with a pioneering method for O-sulfation, a crucial post-translational modification and biotransformation within the metabolism of bioactive molecules.The introduction begins by emphasizing the significance of O-sulfation in various biomolecules and highlights the challenges in their synthesis.Traditional methods, such as sulfur trioxide-nitrogen base complexes, face limitations, prompting the need for alternative strategies like early-stage sulfation.The authors introduce dialkyl sulfates, specifically dimethyl sulfate (DMS) and diisopropyl sulfate (DPS), as promising sulfate diesters for O-sulfation.The proposed method involves activating DMS or DPS under tetrabutylammonium bisulfate, enhancing their reactivity for efficient sulfate transfer.Extensive optimization studies identified DMS as the optimal sulfate source, coupled with Bu4NHSO4 as the ideal activation agent.Control experiments validated the necessity of both DMS and Bu4NHSO4 for successful sulfation.The method demonstrates versatility, effectively sulfating a range of primary and secondary alcohols with DMS and DPS, respectively.The authors showcase the broad applicability of their method to various compounds, including primary alcohols, secondary alcohols, amides, halides, nitrates, ethers, alkenes, alkynes, boronic esters, aldehydes, sulfones, esters, ketones, sulfonamides, aromatics, and heterocycles.Additionally, the method proves effective for the gram-scale synthesis of sulfated products.The paper extends the method's application to carbohydrates, amino acids, and steroids.Various sugar derivatives, amino acids, and complex natural products were successfully sulfated under mild conditions, showcasing the method's practicality for late-stage modifications of bioactive molecules.Results from control experiments provide insights into the reaction mechanism, indicating the involvement of key intermediates such as methyl monosulfates and methyl sulfate D'.Notably, the review includes 18Olabeling experiments, confirming the formation of O-SO3 bonds in the sulfation process.Of note, the manuscript is accompanied by extensive supplementary information (122 pages) In conclusion, the paper preents the discovery of a novel activation method for dimethyl sulfate and diisopropyl sulfate as a significant advancement in the synthesis of organic sulfates.The method's mild reaction conditions, broad functional group tolerance, and applicability to complex biomolecules position it as a powerful tool for late-stage sulfation and drug discovery.The authors anticipate the widespread use of this O-sulfation method in comprehending biological functions and advancing drug discovery.We thank the reviewer for their careful appraisal of our work.
There are no major problems in the manuscript, just several minor comments can be found in the annotated manuscript and supplementary file.We are grateful to the reviewer for their corrections on our typographical errors, and all the suggestions for improving our manuscript.
Reviewer 3 (Remarks to the Author): We thank the reviewer for the time taken to consider our manuscript.We agree with all comments made by this reviewer and present here the changes we made to the revised submission.
The manuscript by Yue et al. represents a noteworthy progression in the field of O-sulfation chemistry.Notably, the authors have elucidated a novel reactivity exhibited by dimethyl sulfate and diisopropyl sulfate, serving as innovative O-sulfation reagents under acidic conditions.The transformations outlined predominantly involve the O-sulfation of alcohols, phenols, and carbohydrates, resulting in the production of sulfonated chemicals.This research is particularly intriguing as it refines the reactivity of dimethyl sulfate and diisopropyl sulfate, traditionally acknowledged for their robust alkylating properties, by harnessing their potential as sulfation reagents.The authors demonstrate the method work with a broad array of electronically and sterically diverse alcohols, carbohydrates, amino acids, and natural products, especially for showcasing selectivity on substrates (55-58).It is noteworthy, however, that the method exhibits limitations in the sulfation of tertiary alcohols and phenols when employing DMS or DPS as the reagents.The authors, in an attempt to address the sulfation of phenols, utilized sodium pyrosulfate as a reagent, a tangent that is not directly pertinent to the focus of this manuscript.Nevertheless, this limitation does not diminish the significance of the work, as the manuscript furnishes compelling evidence that dimethyl sulfate and diisopropyl sulfate can indeed serve as O-sulfation reagents for alcohols and carbohydrates, exhibiting a novel reactivity.We thank the reviewer for their careful appraisal of our work.
The elucidation of how the reactivity of dimethyl sulfate was modulated presents an intriguing aspect of this study.While the paper does furnish some evidence for the proposed pathway, there remain substantial mechanistic inquiries that warrant attention, particularly concerning the substantiation of the reactive intermediate.Several mechanistic hypotheses pivot on the existence of methyl monosulfates (1-B).As it currently stands, additional experiments are imperative to offer more compelling evidence regarding its presence.Given the substantial emphasis on mechanistic elucidation within this manuscript, these unresolved issues pose impediments to recommending it for publication.We agree with the reviewer's concern on this point.Please see our reply to the comments 5-9 on the mechanistic elucidation.
In summary, this work presented by Li and their colleagues represents an important contribution to the field, showcasing a significant advancement in O-sulfation chemistry.The broad substrate scope and excellent selectivity observed in this study open up new avenues for the synthesis of complex and valuable molecules.The compounds are appropriately characterized, and manuscript was well-written.In my opinion, the manuscript merits acceptance for publication in Nature Communications, pending the authors' careful attention to the following suggestions raised.We thank the reviewer for this comment.
Conditions optimization: 1.Given the author's emphasis on the importance of acidic conditions in this reaction, have the authors attempted to incorporate the corresponding acid for entries 5-6 in Table 1?We thank the reviewer for this suggestion.The combined use of Bu4NBF4 and KHSO4 was observed to be more effective than the sole use of Bu4NBF4 (entries1, 2), thereby providing evidence for the crucial role of acidic conditions.A similar effect can also be observed for Bu4OAca and Bu4I (entries 3-6).The presence of the acid HOAc (entry 7), which is more soluble in MeCN compared to KHSO4, causes the decomposition of the sulfate product 1.Additionally, side products such as methyl acetate and acetated-1 derivatives are formed, resulting in a lower overall yield.
We have updated these data in revised Supporting Information (page 5).
2. On the other hand, the yield with a counter anion other than HSO4 is quite good for entries 4-6, especially for entry 5, even in the absence of acid.Does the author have an explanation for how the product was formed in this case?We appreciate the reviewer's meticulous analysis of this aspect.The potential release of conjugated acid (HX) from the equilibrium between the substrate ROH and activator Bu4X (X ≠ HSO4 -) may indeed promote sulfation.This hypothesis can be verified by the decreased pH over time for the solution of 1a and Bu4OAc.

These results can be found in revised Supporting Information (page 6):
In the revised manuscript, we have added the discussion: "The use of Bu4NBF4, Bu4NOAc and Bu4NI as additive that could potentially release their conjugated acid from the equilibrium with 1a, resulted in lower yield." Substrates scope: 3.All the products were well-characterized; however, only NMR yield was provided for substrates (11, 19-24, 58, 61, 65, 66).Addressed.Please see our reply to comment 1 (on SI) of reviewer 1. 2, several other counter cations like Na+, K+, were used.How did the authors determine that these were not included in the final products?We thank the reviewer for this concern.In our case, organic sulfates with different cations have different polarity.We choose compound 1 as an example, and it is evident that they are well separated on the TLC plate.Compared to Na + and K + , the soft Bu4N + cation are considered to be more matched to organic sulfate anion, and their corresponding sulfates exhibit better solubility.

For conditions d in Table
Mechanistic studies: 5.In Figure 2 A, the 1H NMR spectrum of methyl monosulfate (1-B) does not appear consistent with it being the main intermediate, especially given its low integration.Without integrations of the key signals, it is hard to discern the correlation between the new peaks in the 1H NMR and that they correlate to the main intermediate.Integration of these peaks in relation to the standard in 1H spectra would be helpful.We agree that it would be appropriate to give the integration of the intermediates.The new data is added into Figure 2A in the revised manuscript.
In the revised manuscript, we add the discussion about the mechanism: "As the reaction progressed, these intermediate species were converted to some extent into the final sulfate product."6.According to the proposed mechanism in Figure 1C-ii, Bu4NHSO4 would react with intermediate B. Why wouldn't it react with DMS at the beginning, given that DMS is less sterically hindered than intermediate B?
We agree with the reviewer, and have corrected/removed the inappropriate expression in Figure 1C-ii.ASU2 mechanism starts with the activation of DMS in the presence of Bu4NHSO4, we have corrected it in the revised manuscript in Fuire 1C-ii: 7. Have the authors investigated the formation of SO3 during the reaction?They could attempt to detect and capture SO3 by mixing the DMS and Bu4NHSO4.This would enhance the understanding of the reaction mechanism.Addressed.Please see our reply to comment 15 of review 1. 8. Determining the source of the 'sulfur' in the product is crucial, and conducting experiments with 34S labeled Bu4NHSO4 and/or DMS would provide valuable insights.We agree with the reviewer that establishing the source of sulfate in the product is essential for gaining mechanistic insights.The 34 S-labeling experiment provides direct evidence, but synthesizing 34 S-labeled Bu4NHSO4 and DMS from H2 34 S or 34 S is challenging due to their high cost and limited availability.Here, we present two experiments to support the sulfate originating from both of DMS/DPS, and Bu4NHSO4.First, the yield of sulfate product 1 exceeds the stoichiometric amount of any possible sulfur source.Second, DMS reacts with Bu4NHSO4 to yield methyl sulfates mixture (D and D′), which could deliver the final organic sulfates.
In the revised manuscript, we add the following discussion: " The higher yield of 1, in comparison to the stoichiometric amount of any individual sulfur source, suggests that the sulfate group in product originates from both DMS/DPS, and Bu4NHSO4 (Figure 2C)." 9.In drawing the mechanism (Figure 1C-ii, from A to B), based on the arrows the authors have depicted, it suggests the formation of a methoxide anion.However, considering the acidic conditions, the existence of a methoxide anion is improbable.The arrow might be better represented as originating from the oxygen of the alcohol rather than the hydrogen.
O enrichment." "Given the formation of S-SO3 products 20 rather than O-SO3, the intact C-S bond demonstrated that the sulfation of R-OH proceeds via the formation of O-SO3 bond rather than C-OSO3, as the C-S bond is much weaker than C-O.Most importantly, the stereoselective retention of various chiral substrates (46-59, 67-69) in Table 3 clearly verifies the formation of O-SO3 bond."The new data, and 18 O MS spectra is added in the revised supplementary information (page 50, 57): 4) 18O MS data eg screenshots vs abundance predictions needed.Addressed.See our reply to the above comment 3. 5) Need a safety statement re DMS and DPS usage.Addressed.Please see our reply to the very beginning comment 2. 6) 19F NMR for 36 has 2 peaks?